Ethanolamine is released to the external medium at increased rates over basal levels when [3H]ethanolamine-labelled glial cells in primary culture, subcultures of astrocytes and O-2A lineage glia or C6 glioma cells are treated with phorbol ester or foetal calf serum. On equilibrium labelling of mixed glial cultures 90-95% of incorporated [3H]ethanolamine was in ethanolamine-containing phospholipids and 2% in the phosphatidylcholine component. On stimulation of glia with phorbol ester or foetal calf serum ethanolamine, and not phosphoethanolamine or choline, was released to the medium suggesting that a phospholipase D-mediated turnover of lipid, perhaps partially linked to protein kinase C, is involved. The results show that cells in primary culture behave similarly to cell lines in that signal transduction pathways involve a stimulated turnover of ethanolamine-phospholipids with the external release of ethanolamine.